Ascosphaera callicarpa, a New Species of Bee-Loving Fungus, with a Key to the Genus for Europe Anja A. Wynns*, Annette B. Jensen, Jørgen Eilenberg Center for Social Evolution, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, Denmark Abstract We studied the bee specialist fungus Ascosphaera in wild solitary bees to investigate the diversity of the genus in nature and the ecology of these fungi with their bee hosts. A new morphologically distinctive species was discovered which also has a unique nrITS sequence. This new species, here named Ascosphaera callicarpa, is common on the larval feces of the solitary bee Chelostoma florisomne which nests in the Phragmites reeds of thatched roofs in Europe. Because collections of Ascosphaera from wild bees are scarce and because little is known about the ecology and distribution of the majority of the species in the genus, a key to the species thus far reported for Europe is included. Citation: Wynns AA, Jensen AB, Eilenberg J (2013) Ascosphaera callicarpa, a New Species of Bee-Loving Fungus, with a Key to the Genus for Europe. PLoS ONE 8(9): e73419. doi:10.1371/journal.pone.0073419 Editor: Vishnu Chaturvedi, California Department of Public Health, United States of America Received August 30, 2012; Accepted July 22, 2013; Published September 25, 2013 Copyright: ß 2013 Wynns et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the University of Copenhagen and the Danish National Research Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction A distinguishing feature of Ascosphaera is the presence of spore balls [8]. A spore ball is a compact aggregation of spores formed Ascosphaera is a genus of 28 species of bee specialist fungi with by groups of asci that are united by a single membrane [12]. The a worldwide distribution in the temperate to tropical regions. membrane surrounding a spore ball disintegrates and only The genus is remarkable for its host and habitat specificity with remnants of it are sometimes observed in mature spore cysts [5]. all species completing their entire life cycle within the nests of Spore balls may contain as few as two to as many as several bees (Apoidea: Anthophila). Ascosphaera was first discovered in hundred ascospores [5,13]. The average number of ascospores per the early 20th century in Europe after A. apis, the type species, spore ball and the persistence of spore balls at maturity are was identified as the causative agent of a brood disease affecting meaningful taxonomic characters. honeybees [1,2]. This brood disease, known as chalkbrood, was Pathogenic Ascosphaera species afflict only the larval stage of bees. later observed in a solitary bee in London [3]. Ascosphaera is Typically diseased larvae die in the larval stage; however, in rare widely known as the chalkbrood fungus, although at least half of occurrences, larvae have been observed to enter pupation before the species lead a saprotrophic rather than pathogenic lifestyle being overcome by the fungus (Wynns pers. obs.). Pathogenic [4,5]. Saprotrophic Ascosphaera species flourish on diverse species of Ascosphaera appear to be highly specialized fungi with substrates within the bee nest, for example on pollen provisions, ascospores typically germinating only when within the midgut of on materials used by the bees to construct the nest and on larval their host. Spore germination is followed by rapid hyphal growth, feces [6,7]. Little is known about these saprotrophs which with the fungus consuming the larva from the inside out [14]. Two appear to live innocuously inside the brood cells of the bees. widespread pathogenic species, Ascosphaera aggregata and A. apis, are Consequently, the potential for research on the ecological and of economic interest because of their potential to negatively impact functional role of these fungi within the bee nest remains wide populations of commercial pollinators, namely Apis mellifera L. and open. Megachile rotundata (Fabricius) [15,16]. Ascosphaera is placed in Ascosphaeraceae (Pezizomycotina: Although Ascosphaera lives in association with both solitary and Eurotiomycetidae), a small family of ascomycetes primarily social bees the majority of species (25 out of 28) were originally characterized by a unique fruiting body type called a spore cyst. described from solitary bees. Within the nests of solitary bees Spore cysts are unicellular, cyst-like fruiting bodies that form from Ascosphaera grows on pollen provisions where an egg has failed to the expansion of a single cell called a nutriocyte [8]. The wall of a develop, on larval feces, on the surface of cocoons, within larvae, spore cyst is a double-layered membrane. Asci are free-floating and on the diverse materials used by different bee species for and evanescent. Because of their anomalous fruiting bodies, the brood cell construction [4,5,6]. Unlike their social relatives (e.g., taxonomic affinities of Ascosphaera and its relatives remained honey bees), solitary bees lack adult-larva interaction, there is no uncertain until ontological studies led C.F. Spiltoir and L.S. Olive nursing of the brood and no cooperative behavior (including social [8] to confidently place them among the Ascomycota within immunity) [17]. A consequence of no adult-larva contact and no Eurotiomycetidae [as Plectascales]. This position was later nursing is that the brood is mass provisioned rather than confirmed by additional morphological study [9] and DNA progressively provisioned like their social counterparts; this means sequenced-based phylogenies [10,11]. that once an egg hatches the larva has all the food it will need to PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e73419 Ascosphaera callicarpa sp. nov. complete development into an adult [17]. Following their flight (Qiagen) and sent to Eurofins MWG Operon AG (Ebersberg, and nesting period solitary bees overwinter in their individual Germany) for sequencing. The nucleotide sequence was assembled brood cells with no activity until emergence the following spring or using BioEdit [22] and subjected to a BLASTn search in early summer. In this way solitary bee nests provide a relatively GenBank. stable, undisturbed micro-environment that appears suitable for the growth of these specialised fungi. Nomenclature The only monographic work on Ascosphaera [5] focused on The electronic version of this article in Portable Document collections from an important commercial pollinator in Canada, Format (PDF) in a work with an ISSN or ISBN will represent a the alfalfa leafcutting bee Megachile rotundata. While limited in published work according to the International Code of Nomen- scope, this monograph, which included the first key to the genus, clature for algae, fungi, and plants, and hence the new names remains the most useful and comprehensive reference for the contained in the electronic publication of a PLOS ONE article are identification of Ascosphaera species. Given the importance of wild effectively published under that Code from the electronic edition pollinators and their increasing role in buffering the loss of alone, so there is no longer any need to provide printed copies. In honeybee pollination services [18] a more complete monograph addition, new names contained in this work have been submitted with an updated key to these bee-specialist fungi is much needed. to MycoBank from where they will be made available to the Seven of the 28 described species of Ascosphaera are currently Global Names Index. The unique MycoBank number can be known from Europe. Here we describe a new species from resolved and the associated information viewed through any Denmark occurring in the nests of the wild solitary bee Chelostoma standard web browser by appending the MycoBank number florisomne L. To stimulate interest and to facilitate the identification contained in this publication to the prefix http://www.mycobank. of Ascosphaera species so far known from Europe, we provide a key org/MB/. The online version of this work is archived and and descriptions for these species. Cumulative host reports and available from the following digital repositories: PubMed Central, species distributions are also included with the hope that this LOCKSS. information will result in additional records for these under- collected fungi. Results and Discussion Materials and Methods Culture and isolation Despite repeated attempts, we were unable to obtain in vitro Morphological study mycelial growth or induce ascospore germination of Ascosphaera Descriptions of spore cysts and ascospores were made from callicarpa. observations of spore cysts mounted in water on a glass slide. Measurements and light photomicrographs were made on an Molecular study Olympus AX70 Provis light microscope and Olympus SZX16 An ITS sequence was obtained for A. callicarpa (GenBank dissecting microscope. Herbarium acronyms follow those of Index accession: JX070046). A BLASTn search of the ITS sequence Herbariorum [19]. revealed a highest sequence-similarity to other Ascosphaera species. Culture and isolation Key to European species of Ascosphaera Attempts to